Calmodulin (CaM) regulation of four-domain channels first emerged in CaV channels, and has proved rich both mechanistically and biologically. Our laboratory has been one of the leaders in unveiling this exciting chapter of CaV channel discovery. Given the sequence similarity of CaV and NaV channels, intriguing suspicions arose that NaV channels might also exhibit such CaM regulation. This possibility was most attractive, given the wide-ranging biological and clinical impact of NaV channels, which include mentation (epilepsy), muscle contraction (myotonia and arrhythmias), and sensation (neuropathic pain). Biochemistry and structural biology both underscored this similarity, but the reported functional effects of Ca2+ and/or CaM on NaV channels have been rather subtle, mostly limited to several-mV shifts of steady-state inactivation (h%) curves. Yet, channelopathic NaV mutations at putative structural determinants of Ca2+ and/or CaM regulation do confer severe disease phenotypes. One potential culprit for the apparent disconnect is that, unlike CaV channels, NaV channels do not conduct Ca2+, so they cannot directly trigger Ca2+ responses. Instead, the NaV field has used whole-cell dialysis to tonically manipulate Ca2+ levels, over several minutes or longer. Could the field be characterizing desensitized responses to Ca2+ and/or CaM, with poor similarity to short-term and larger physiological effects? Here, we use a different approach to dynamically perturb Ca2+, and our preliminary data unveil something long sought in the field>rapid and robust Ca2+/CaM-dependent inactivation of NaV channels (CDI). The advances now permitted may revolutionize understanding of the CaM regulation of NaV channels. This project will usher in an exciting era of discovery via three specific aims. (1) To unveil the existence of rapid Ca2+/CaM-mediated regulation across the family of NaV channels. (2) To elucidate the mechanism of dynamic CaM-mediated regulation of NaV channels. With long-sought robust readouts of NaV regulation in hand, Aim 2 will be uniquely poised to dissect the mechanistic underpinnings of regulation. (3) To assess the broader biological impact of CaM-mediated regulation of Nav channels. The mechanistic advances above hold numerous biological implications that Aim 3 will explore. In all, this project will facilitate a period of unprecedented progress in the Ca2+ regulation of NaV channels. As well, this proposal will explicitly link Ca2+ regulation of NaV channels to certain channelopathies, and perhaps to related but more generalized forms of disease.